Project 8

Background and preliminary work: Despite its limited regenerative capacity, the adult heart demonstrates remarkable plasticity. Physiological hypertrophy can adapt ventricular cardiac function to increased mechanical stress, whereas disease-related stress, such as chronic hypertension or myocardial injury, induces maladaptive hypertrophic growth, which increases the risk of functional decompensation and heart failure. Myocardial plasticity also allows more rapid adjustments to ventricular function in order to respond to acute changes, such as myocardial ischemia. This includes dynamic adaptation of the passive stiffness of the myocardium, which modulates cardiac function by determining the distensibility of the ventricles during diastolic filling. The elastic properties of the myocardium are defined by the compliance of both the connective tissue and the cardiomyocytes. In cardiomyocytes, Ca²⁺-independent (passive) stiffness is largely determined by the giant sarcomere protein titin and can be modified via posttranslational modification and altered isoform composition ^{1, 2}. In response to myocardial injury as well as in the progression of manifest heart failure, changes in connective tissue and extracellular matrix are closely related to adaptations in the passive elastic properties of cardiomyocytes and altogether mediate a complex sequence of temporally and spatially regulated adaptation and long-term remodeling of the myocardium ³.

However, we previously demonstrated that in response to myocardial infarction, the integrity of the surviving (remote) myocardium is maintained by an acute increase in passive stiffness of CM that precedes interstitial fibrosis ⁴ and later regresses with the onset of fibrotic scar formation. This observation indicates that the molecular plasticity of CMs also allows more dynamic adaptation of ventricular function. Given the structural and functional interdependence of CMs and FB in the heart, the functional adaptations of both cell types in response to mechanical stimuli are likely interrelated. The precise molecular mechanisms that mediate a rapid and dynamic interplay between CM properties, extracellular matrix and FB are not fully understood. 

Our group previously described that the pro-inflammatory cytokine IL-6, which induces PKCα-dependent phosphorylation of titin, contributes to the increased cardiomyocyte passive stiffness after myocardial ischemia/reperfusion ^{5, 6}. More recently, we found that TGF-β modifies titin-based passive and active properties of cardiomyocytes by altering its phosphorylation state and by shifting titin isoform composition towards the stiffer N2B variant (unpublished data). Cardiac FB are an important source and target of IL-6 mediated signaling in the heart, and activation of cardiac FB is mediated by paracrine factors such as TGF-β, angiotensin II, but also by mechanical stress. These findings indicate that pro-inflammatory signaling pathways may serve as novel regulators of passive stiffness mediated by CMs and FBs during myocardial remodeling. 

We are confident that studying the mechanical behavior and interaction of cardiac cell populations in vitro and in vivo will significantly enhance our understanding of early myocardial remodeling. To this end, we have joined forces with Prof. Steven Caliari’s group, which has expertise in the design, synthesis and characterization of new biomaterials for tissue engineering. They have developed a range of matrices with defined, scalable stiffness for investigating the dynamic interplay between cells (myoblasts, hepatic stellate cells, fibroblasts, etc.) and their microenvironment ^{7, 8}. They demonstrated that in vitro, FBs rapidly respond to stiffening of their surrounding substrate by changing their cell shape, DNA methylation, and chromatin organization, suggesting cellular reprogramming ⁹. 

Hypothesis: We hypothesize that changes in cardiomyocyte (CM) stiffness and cardiac fibroblasts (FB) activation are functionally linked and that mechanosensitive responses such as release of proinflammatory cytokines, may be crucial for myocardial plasticity and are triggers for phenotype changes in neighboring CM and FB.

WP 1: A preliminary assessment of the state of CM and FB in the non-ischemic myocardium will be conducted by comprehensively characterizing the FB differentiation and activation state and the morphological changes of CM in the non-ischemic heart after infarction. Cells will be isolated from non-ischemic myocardium of mice 24h and 72h after I/R induced by 45 min. ligation of the left anterior descending artery ⁵. CM and FB will be separated by MACS purification ¹⁰. RNAseq and bioinformatic analysis will be performed to analyze cell-type specific changes in transcriptional regulation. With this unbiased approach, we expect to identify novel signaling pathways and candidate genes possibly involved in the early transmission of mechanosensitive processes. Effects of selected pathways or genes on CM and FB will be further analyzed in WP2. In collaboration with project 7, we will compare our results with data derived from spatial transcriptomic analysis of cardiac tissue slices. To test for I/R-induced activation of inflammatory processes in the non-ischemic myocardium, cytokine concentrations will be determined at different time points after I/R in tissue lysates using multiplex cytokine assays and ELISA.

WP 2: We will further analyze cultivated CMs and FBs and its acute modulation in response to different substrate stiffnesses. Initial experiments planned in this work package will be carried out during the exchange phase at UVA, as we will take advantage of different culture systems with tunable substrate stiffness, previously established by the Caliari group. Previous studies indicate that healthy myocardium has a stiffness (Young’s modulus) of 10–18 kPa, while diseased myocardium can stiffen beyond 50 kPa ¹¹. Therefore, isolated adult rat CMs and FBs will be cultured separately for 2 hours up to 3 days on substrates with adjustable stiffness, ranging from 1 to ~50 kPa.

A subset of cultured CM and FB will be stimulated with conditioned medium from the other cell type to test whether changes in substrate stiffness lead to the release of signaling molecules that influence the morphology and function of other cells. Another subset of cultured cells will be challenged by application of proinflammatory stimuli (e.g. IL-6 and TGF-β), or compounds affecting signaling pathways or genes identified in WP1. 

For all experimental conditions, microscopic analyses will be performed at different time points after onset of treatment to study changes in the cellular and subcellular morphology such as cellular dimensions, activation state (FB), myofibrillar density, sarcomere and myofilament length, microtubule dynamics, costamere formation. Mass spectrometry (BMFZ core facility at HHU) will be used to characterize the acute influence of substrate stiffness on posttranslational modification (e.g., phosphorylation, S-glutathionylation, acetylation) of key components involved in mechanotransduction and data will be confirmed using Western blot technique. Multiplex assays (and mass spectrometry) will be performed to test for changes in the secretome of CMs and FBs. Within the first 24 hours of CM culture, it is possible to detach CMs from the culture plate and use them for functional force measurements (unpublished). Thus in a subset of CMs, Ca²⁺-induced isometric contractile force development, activation/relaxation kinetics, and passive tension will be determined with a force transducer and a piezo-stimulated length controller (myocyte workstation, Aurora Scientific). 

WP 3: The functional relevance of findings from the other two work packages will be tested in isolated rat papillary muscles. These can be easily isolated from hearts and can be considered as a 3D co-culture of CMs and FBs with functional cell-cell contacts and the possibility of short-term culture (up to 6h). Contraction of papillary muscles can be evoked by electrical stimulation and the effect of cytokines, or modifiers of mechanotransduction (see work package 2) on the contractile behavior can be monitored using a mechanical workstation (MyoDYNAMIC Muscle Strip System 840 MD, DMT). To mimic different levels of mechanical stress after I/R, papillary muscles can be longitudinally pre-stretched. We hypothesize that the short-term application of these different stimuli is sufficient to challenge the short-term plasticity of CM and FBs and induce phenotypic changes that will influence the contractile behavior of the papillary muscle. Biochemical analysis will be performed to detect changes in expression, degradation or modification of proteins involved in CM and FB mechanotransduction. In a subset of stretch experiments conditioned media will be collected and analyzed to identify stretch-induced release of ECM-stored signaling molecules, such as cytokines. 

Added value of collaboration: The planned project will significantly benefit from the combined expertise of the UVA cooperation partner Prof. Steven Caliari in the field of matrix biology and tissue engineering and of our group in exploring myocardial stiffness and titin modulation in response to various pathological conditions. During the exchange phase at UVA, the PhD student from Düsseldorf will gain expertise in the use of different culture systems with tunable substrate stiffness and will identify the best suitable system and the optimal culture conditions to analyze the effects of substrate stiffening or softening on the functional behavior of adult rat cardiomyocytes. The method that was best suited for the investigations will then be established by the student in the home laboratory at HHU. In return, a doctoral student from UVA will learn the biomechanical methods of force measurements on single cells and multicellular preparations in Düsseldorf.